Novel contrast agents

ABSTRACT

This invention includes agents and compositions having MRI, PET, CT, X-ray, SPECT or optical signals, and methods for their use in the determination of a target. In some cases, a MRI, PET, CT, X-ray, SPECT, optical or other signal produced by the agent or composition can be affected by the presence of the target. Examples of targets that can be determined by this invention include, but are not limited to zinc, copper, iron ions and other biological targets. Example of application for imaging in vivo includes the function of pancreas and other organs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/748,445, filed Mar. 28, 2010, which claims priority to U.S.Provisional Application No. 61/225,187 filed Jul. 13, 2009, thedisclosures of which are herein incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of chelatingagents, a method for preparing chelating agents and methods of usingchelating agents in diagnostic and therapeutic applications.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with contrast agents that detect a target in vivo forinstance Zinc.

SUMMARY OF THE INVENTION

In one aspect, this invention relates to contrast agents for MRI, PET,CT, X-ray, SPECT, optical or ultrasound techniques consisting of achelator group comprising a target binding unit, wherein the bindingmoiety is capable of binding a target further binding a protein in vivosuch as human serum albumin and generating a signal that can be detectedby MRI, PET, CT, X-ray, SPECT, optical or ultrasound techniques in vivo.

In another aspect, this invention also relates to compositionsconsisting of metal complexes of the chelator group wherein the metalcomplex has determinable signal by MRI, PET, CT, X-ray, SPECT, opticalor ultrasound techniques wherein the binding target unit is capable ofinteracting with a target and further binding a protein in vive such ashuman serum albumin generating a signal that can be detected MRI, PET,CT, X-ray, SPECT, optical or ultrasound techniques.

The present invention also relates to improved processes for thepreparation of contrast agents consisting of a chelator group having atarget binding unit.

Aspects of this invention include a MRI, PET, CT, X-ray, SPECT, opticalor ultrasound agent or composition, having: a chelator group, a bindingmoiety covalently attached to the chelator group, wherein the bindingmoiety is capable of binding a target such that MRI, PET, CT, X-ray,SPECT, optical or ultrasound signal is changed upon binding the target,wherein the chelator group optionally comprises a metal, and wherein theagent is capable of detecting a target in vive in a subject.

In some embodiments, the chelator group can include a macrocycle groupcontaining at least nine atoms which at least three being heteroatoms;and the binding moiety can be N,N′-bis-2-pyridyl methyl amine (BPMA).

Yet in another aspect, the metal ion can be at least one paramagneticmetal such as Gd³⁺, Eu³⁺, Tm³⁺, Dy³⁺, Tb³⁺, Yb³⁺, Mn²⁺, Ce, Pr, Nd, Pm,Sm, Ho, Er, Lu and Y. A radioactive metal such as ¹¹¹In³⁺, ^(113m)In³⁺,⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺, ⁶⁴Cu²⁺, Tl³⁺, ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺ and ²¹²Bi³⁺,⁹⁰Y³⁺, ³⁷⁷Lu³⁺, ²²⁵Ac³⁺, ¹⁴⁹Pm, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re,⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu, ¹⁹⁸Au, ^(117m)Sn, and ²¹²Pb.

In some aspects, this invention includes an agent having the formula:

wherein X₁, X₂, X₃ and X₄ can be the same or different and each is aheteroatom preferably nitrogen.

In some aspects, M can be absent or if present M is a metal ion whichcan be used in MRI, PET, SPECT and optical, depending on therelaxometric, luminescent, fluorescent, or radioactive properties of thefinal form of the metal chelate contrast agent before and after bindingto the relevant target. For example for diagnostic by MRI Gd³⁺, Eu³⁺,Tm³⁺, Dy³⁺, Tb³⁺, Yb³⁺ and Mn²⁺ are preferred. For radiodiagnostic¹¹¹In³⁺, ^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺, ⁶⁴Cu²⁺, Tl³⁺, or, forradiotherapy ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺ and ²¹²Bi³⁺, and more preferably, ⁹⁰Y³⁺,³⁷⁷Lu³⁺ and ²²⁵Ac³⁺. Other metal ions in some embodiments forradiodiagnostic or radiotherapeutic applications include, but are notlimited to: ¹⁴⁹Pm, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁷Ru, ¹⁰⁵Rh,¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu, ¹⁹⁸Au, ^(117m)Sn, and ²¹²Pb. Other metals can be Ce,Pr, Nd, Pm, Sm, Ho, Er, Lu or Y. n₁, n₂, n₃ and n₄ can be the same ordifferent and each indicates a number of methylene (CH₂) units withinthe heterocyclic structure. These can be one, two, three or greater butpreferably one.

Yet in other aspects, R₁ and R₂ can be the same or different and each isan acetate group, optionally this can be substituted in the methylenegroup. Alternatively, R₁ and R₂ can be an acetyl amide group whichnitrogen can be substituted by an alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,aryl or heterocycle unit optionally substituted. Alternatively, R₁ andR₂ can be an acetyl ester where the oxy substituent can be alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, aryl or heterocycle unit optionallysubstituted. Alternatively, R₁ and R₂ can include an organic compoundhaving binding affinity for a cell surface receptor. Examples of suchunits comprise peptides, peptoids, or antibodies.

In some aspects, R₃ indicates a substituent or substituents in themethylene units of the macrocycle. These substituents can be of anychemical nature as described herein and if several they can be the sameor different. A₁ and A₂ can be the same or different and each indicatesan acetyl group preferably, optionally this can be substituted in themethylene group. H₁ and H₂ can be the same or different and eachindicates a heteroatom preferably nitrogen. S₁ and S₂ can be the same ordifferent and each indicates an alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,aryl or heterocycle unit optionally substituted. Alternatively, S₁ andS₂ might not be present. E₁ and E₂ can be the same or different and eachindicates a heteroatom preferably nitrogen. Alternatively, E₁ and E₂might not be present. L₁, L₂, L₃ and L₄ can be the same or different andeach comprises a target binding moiety which upon binding the target canbind another molecule further for example a protein. L₁, L₂, L₃ and L₄can be a substituted derivative of a heterocycle unit such as but notlimited to pyridine, pyrazole, imidazole, piperidine, pyrazine,pyrimidine, pyrrol. Alternatively, L₁, L₂, L₃ and L₄ can be an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl or aryl optionally substituted.

In one aspect, the agent has the formula:

where B includes a cyclic compound within the macrocycle including oneheteroatom of the macrocycle. This cyclic moiety can be aliphatic oraromatic with or without heteroatoms other than the one in themacrocycle, optionally the cycle might be substituted.

Yet in another aspect, agent has the formula:

where C includes a cyclic compound within the macrocycle. This cyclicmoiety can be aliphatic or aromatic with or without heteroatoms,optionally substituted.

Yet in another aspect, agent has the formula:

wherein: X₁, X₂, and X₃ can be the same or different and each is aheteroatom nitrogen. M can be absent or if present M is a metal ionwhich could be used in MRI, PET, SPECT or optical, depending on therelaxometric, luminescent, fluorescent, or radioactive properties of thefinal form of the metal chelate contrast agent before and after bindingto the relevant target. For example for diagnostic by MRI Gd³⁺, Eu³⁺,Tm³⁺, Dy³⁺, Tb³⁺, Yb³⁺ or Mn²⁺. For radiodiagnostic ¹¹¹In³⁺,^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺, ⁶⁴Cu²⁺, Tl³⁺, or, forradiotherapy ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺ and ²¹²Bi³⁺, and, ⁹⁰Y³⁺, ³⁷⁷Lu³⁺ and²²⁵Ac³⁺. Other metal ions for radiodiagnostic or radiotherapeuticapplications include, but are not limited to: ¹⁴⁹Pm, ¹⁵⁹Gd, ¹⁴⁰La,¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu, ¹⁹⁸Au,^(117m)Sn, or ²¹²Pb. Other metals can be Ce, Pr, Nd, Pm, Sm, Ho, Er, Luor Y. n₁, n₂, and n₃ can be the same or different and each indicates anumber of methylene (CH₂) units within the heterocyclic structure. Thesecan be one, two, three or greater but preferably one. R₁ is an acetategroup, optionally this can be substituted in the methylene group.Alternatively, R₁ can be an acetyl amide group which nitrogen can besubstituted by an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl orheterocycle unit optionally substituted. Alternatively, R₁ can be anacetyl ester where the oxy substituent can be alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, aryl or heterocycle unit optionally substituted.Alternatively, R₁ can comprise an organic compound having bindingaffinity for a cell surface receptor. Examples of such units comprisepeptides, peptoids, or antibodies. R₃ indicates a substituent orsubstituents in the methylene units of the macrocycle. Thesesubstituents can be of any chemical nature as described herein and ifseveral they can be the same or different A₁ and A₂ can be the same ordifferent and each indicates an acetyl group preferably, optionally thiscan be substituted in the methylene group. H₁ and H₂ can be the same ordifferent and each indicates a heteroatom preferably nitrogen. S₁ and S₂can be the same or different and each indicates an alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, aryl or heterocycle unit optionallysubstituted. Alternatively, S₁ and S₂ might not be present. E₁ and E₂can be the same or different and each indicates a heteroatom preferablynitrogen. Alternatively, E₁ and E₂ might not be present. L₁, L₂, L₃ andL₄ can be the same or different and each comprises a target bindingmoiety which upon binding the target can bind another molecule furtherfor example a protein. L₁, L₂, L₃ and L₄ can be a substituted derivativeof a heterocycle unit such as but not limited to pyridine, pyrazole,imidazole, piperidine, pyrazine, pyrimidine, pyrrol. Alternatively, L₁,L₂, L₃ and L₄ can be an alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl oraryl optionally substituted.

In some aspects, the agent of this invention can have the structure:

where in: M is Gd³⁺, Eu³⁺, Tm³⁺, Dy³⁺, Tb³⁺, Yb³⁺, Mn²⁺, Ce, Pr, Nd, Pm,Sm, Ho, Er, Lu or Y. At least one radioactive metal such as ¹¹¹In³⁺,^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺, ⁶⁴Cu²⁺, Tl³⁺, ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺and ²¹²Bi³⁺, ⁹⁰Y³⁺, ³⁷⁷Lu³⁺ and ²²⁵Ac³⁺, ¹⁴⁹Pm, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁷⁵Yb,⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu, ¹⁹⁸Au, ^(117m)Sn,or ²¹²Pb.

In one aspect, the target is a metal ion for instance Zn²⁺ or Cu²⁺ ormacromolecular receptor such as a protein.

Yet in another aspect, this invention include a process for making anagent as where the chelator unit is covalently attached to the bindingunit with a coupling agent and a base, wherein said coupling agent canbe an uronium-containing compound or a phosphonic acid cyclic anhydridefor instance wherein said can beO-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate or1-propanephosphonic cyclic anhydride. The method can further includecleaving said of ester groups in presence of an acid to form carboxylicacids, wherein said acid can be hydrochloric acid or trifluoroaceticacid.

In one aspect, this invention includes a method of generating images ofan animate human or non-human subject involving administering a contrastagent to said subject and generating an image of at least a part of thesaid subject to which said contrast agent has distributed, accumulatedand or bound to the target, characterized in that as said contrast agentis used as matter of formula. In one aspect, the image is a magneticresonance image (MRI), a nuclear imaging image, an ultrasound image, oran optical image.

In some aspects, the levels of a target in a given image can bedetermined by administering a low concentration of the agent orcomposition such as in the micromolar range for MRI and nanomolar orlower with other nuclear imaging techniques that involvingradionuclides, and that the concentration of the agent or composition issuch that its signal in an image is zero/baseline background withoutsignificant target concentration.

Yet in another aspect, this invention includes a pharmaceuticalcomposition having a composition of matter of formula together with atleast one pharmaceutically effective carrier or excipient.

Yet in another aspect, the present invention include a method of makingthe novel composition comprising attaching the chelator unit covalentlyto the binding unit using a coupling agent and with a base dissolved ina solvent, wherein the coupling agent comprises a uronium-containingcompound, and wherein said solvent comprises polar aprotic solvents; andtreating the resultant purified product with an acid to cleave estergroups as in claim 4 to form carboxylic acids. The coupling agentcomprises a phosphonic acid cyclic anhydride; and wherein said bases arenon-nucleophilic organic tertiary amines or non-nucleophilic inorganicbases.

In another aspect, the present invention includes a method of generatingimages of an animate human or non-human subject comprising administeringa contrast agent to said subject; and generating an image of at least apart of said subject to which said contrast agent has distributed,accumulated or bound to the target wherein said image is a magneticresonance (MRI), nuclear, ultrasound or optical image.

In another aspect, the present novel composition can be used as apharmaceutical composition comprising the composition of matter offormula as defined in claim 1 together with at least onepharmaceutically effective carrier or excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows a general formula for a class of tetraheterocyclic-basedagents, according to some embodiments of the invention;

FIG. 2 shows a general formula for a class of triheterocyclic-basedagents, according to some embodiments of the invention;

FIG. 3 shows a general formula for a class of tetraheterocyclic-basedagents containing an external cyclic moeity, according to someembodiments of the invention;

FIG. 4 shows a general formula for a class of tetraheterocyclic-basedagents containing an external cyclic moeity, according to someembodiments of the invention;

FIG. 5 shows a tetraazaheterocyclic agent named herein DOTA-diBPMA thatcan be useful in the determination of a target for example Zn²⁺ by MRI,PET, CT, X-ray, SPECT or optical apparatus depending on M;

FIG. 6 shows the synthesis of an agent that comprises as a chelator a1,4,7,10-tetraazacyclododecane unit substituted in the nitrogen atomswith acetate and acetyl units which is then bound to a target bindingunit named herein BPMA, according to one embodiment of the invention;

FIG. 7 shows a graph of the relaxivity of GdDOTA-diBPMA in 0.1 M Trisbuffer pH 7.6 in presence and absence of different metal ions (M).Measurements done at 37° C., pH 7.6 0.1M Tris buffer, 23 MHz;

FIG. 8 shows a graph of the relaxivity of GdDOTA-diBPMA in 0.1 M Trisbuffer pH 7.6 with human serum albumin 0.6 mM in presence and absence ofdifferent metal ions (M). Measurements done at 37° C., pH 7.6 0.1M Trisbuffer, 23 MHz;

FIG. 9 shows a titration of GdDOTA-diBPEN 1 mM (triangles), andGdDOTA-diBPMA 1 mM plus 2 mM of Zn²⁺ (squares) with HSA, the measuredvariable is an enhancement relaxation rate factor (ε*) which representsthe ratio of the difference in relaxation rates of the contrast agent inbuffer with HSA minus the relaxation rate of buffer HSA and therelaxation rate of the agent in buffer minus the relaxation rate of thebuffer;

FIG. 10 shows a T1-weighted phantom MR images. Images of a 100 μMsolution of GdDOTA-diBPMA in Tris buffer with 600 μM HSA at variousconcentrations of ZnCl₂. Spot A, 200 μM Zn; B, 100 μM Zn; C, 30 μM Zn;D, 0 μM Zn; E, no contrast agent and F, Tris buffer without HSA andwithout contrast agent Repetition time (TR)=200.0 ms; echo time (TE)=8.3ms; data matrix=128×128. FOV=15×15 mm². Single scan, no averaging. Asingle slice of 5 mm was acquired centered at the sample height (10 mm);

FIG. 11 shows in A) a post-mortem photograph of a mouse after midlinelaparotomy, the head of the mouse is in the top of the figure and thetail is in the bottom, the pancreas is delineated by the dotted line andpositioned by the long arrow-bead lines. The stomach and liver positionis indicated by the small arrow-head line. In B) the difference MR Imageof the corresponding alive glucose stimulated mouse before injection ofGd³¹DOTAdiBPMA and after three minutes from the injection. The siteswith enhancements due to zinc binding are shown as bright spots. In C) aproton-density with slight T1-weighted MR image slice of the pancreas ofthe corresponding mouse thoracic area is shown to highlight anatomicalfeatures. In D) the overlay of B and C is shown.

FIG. 12 shows PET images (overlayed on CT scans) of mice with highglucose (i.p. injected with 20% glucose solution (2 g/kg body weight)(mouse 1, A) and low glucose (saline only) (mouse 2, B) after beinginjected with ⁶⁴Cu²⁺ DOTAdiBPMA. Note the absence of the pancreas isletsignals in the low glucose compared to the high glucose mouse (whitearrows).

Other aspects, embodiments and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patentsincorporated herein by reference are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example can be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, the term in vive describes living organisms such asanimals and humans.

As used herein, “signal shift” refers to an alteration on the detectedsignal obtained by MRI, PET, SPECT, optical and ultrasound apparatusgiven the presence of an agent or composition, the specific binding ofthe agent to a target and further binding to a macromolecule. Examplesare changes in the relaxometric, luminescent, fluorescent, orradioactive properties of the agent which translate in changes on signalintensities of the detected signals by MRI, PET, SPECT, optical andultrasound techniques.

As used herein the term “determining” generally refers to the analysisof a species or signal, for example quantitatively or qualitatively,and/or the detection of the presence or absence of the species signals.“Determining” can also refer to the analysis of an interaction betweentwo or more species or signals.

As used herein, the term “paramagnetic metal ion” describes a metal ionhaving unpaired electrons, causing the metal ion to have a measurablemagnetic moment in the presence of an externally applied field. Examplesof suitable paramagnetic metal ions, include, but are not limited to,ions of iron, nickel, manganese, copper, gadolinium, dysprosium,europium, and the like. In some embodiments, an agent or composition ofthis invention, can have a chelator group bound to a paramagnetic metalion, wherein the paramagnetic ion is Gd³⁺.

As used herein, an emission's can be a luminescence emission, in which“luminescence” is defined as an emission of ultraviolet or visibleradiation. Specific types of luminescence include, but are not limitedto, fluorescence, phosphorescence, chemiluminescence,electrochemiluminescence, and the like. In some cases, the emission canbe fluorescence emission.

In the compounds and compositions of the invention, the term “alkyl”refers to the radical of saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In some embodiments, a straight chain orbranched chain alkyl can have 30 or fewer carbon atoms knits backbone,and, in some cases, 20 or fewer e.g., 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. In some embodiments, a straightchain or branched chain alkyl has 12 or fewer carbon atoms in itsbackbone. Yet in another embodiments 6 or fewer, and 4 or fewer.Likewise, cycloalkyls have from 3-10 carbon atoms in their ringstructure, and can have 5, 6 or 7 carbons in the ring structure.Examples of alkyl groups include, but are not limited to, methyl, ethyl,propyl, isopropyl, cyclopropyl, butyl, isobutyl, tell-butyl, cyclobutyl,hexyl, cyclohexyl, and the like.

The term “heteroalkyl” refers to an alkyl group as described herein inwhich one or more carbon atoms is replaced by a heteroatom. Suitableheteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like.Examples of heteroalkyl groups include, but are not limited to, alkoxy,amino, and the like.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contained at least one double or triple bondrespectively.

The terms ‘heteroalkenyl” and ‘heteroalkynyl” refer to unsaturatedaliphatic groups analogous in length and possible substitution to theheteroalkyls described above, but that contain at least one double ortriple bond respectively.

As used herein, the term “halogen” or “halide” designates —F, —Cl, —Br,or —I.

The terms “acetyl group” are recognized in the art and can include suchmoieties as can be represented by the general formula: —C(C═O)G, whereif G is OH the formula represents an acetate; if G is O—P where P can bealkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, aryl or heterocycle theformula represents acetyl esters and N—P where P can be alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, aryl or heterocycle the formula representsacetyl amides, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl orheterocycle are the substituents in the nitrogen or oxy part of theacetyl group.

The term “aryl” refers to aromatic carbocyclic groups, optionallysubstituted, having a single ring (e.g., phenyl), multiple rings (e.g.,biphenyl), or multiple fused rings in which at least one is aromatic(e.g. naphthyl, antryl, or phenanthryl). That is, at least one ring canhave a conjugated pi electron system, while other, adjoining rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles.

The term “macrocycle” refers to cyclic groups containing three or fourheteroatoms in their structure with the remainder of the ring of thering being carbon atoms at least 6.

The term “heterocycle” refers to cyclic groups containing at least oneheteroatom as a ring atom, in some cases, 1, 2 to 3 heteroatoms as ringatoms, with the remainder of the ring atoms being carbon atoms. Suitableheteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like.In some cases, the heterocycle can be 3-, 4-, 5-, 6-, 7-, 8-, 9- to10-membered ring structures. Yet in another embodiment, 3- to 7-memberedrings, whose ring structures include one to four heteroatoms. The term“heterocycle” can include heteroaryl groups (e.g., aromaticheterocycles), saturated heterocyclic (e.g., cycloheteroalkyl) groups,or combinations thereof. The heterocycle can be a saturated molecule, orcan comprise one or more double bonds. In some case, the heterocycle isan aromatic heterocycle, such as pyrrole, pyridine, and the like. Insome cases, the heterocycle can be attached to, or fused to, additionalrings to form a polycyclic group. In some cases, the heterocycle can bepart of a macrocycle. The heterocycle can also be fused to a spirocyclicgroup. In some cases, the heterocycle can be attached to a compound viaa nitrogen or a carbon atom in the ring. Heterocycles include, forexample, thiophene, benzothiophene, furan, tetrahydrofuran, pyran,isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole,dihydropyrrole, pyrrolidine, imidazole, pyrazole, pyrazine, isothiazole,isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline,quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, carbazole, carboline, triazole, tetrazole,oxazole, isoxazole, thiazole, isothiazole, phenanthridine, acridine,pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazine,piperidine, homopiperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, othersaturated and/or unsaturated derivatives thereof, and the like. Theheterocyclic ring can be optionally substituted at one or more positionswith such substituents as described herein. In some cases, theheterocycle can be bonded to a compound via a heteroatom ring atom(e.g., nitrogen). In some cases, the heterocycle can be bonded to acompound via a carbon ring atom.

As used herein the terms “amine” and “amino” describes bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula: N(R′)(R″)(R′″) wherein R′ R″ and R′″each independently represent a group permitted by the rules of valence.

Any of the above groups can be optionally substituted. As used herein,the term “substituted” is contemplated to include all permissiblesubstituents of organic compounds, “permissible” being in the context ofthe chemical rules of valence known to those of ordinary skill in theart. It will be understood that “substituted” also includes that thesubstitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination. In some cases, “substituted” can refer toreplacement of a hydrogen with a substituent as described herein.However, “substituted,” as used herein, does not encompass replacementand/or alteration of a key functional group by which a molecule isidentified, e.g., such that the “substituted” functional group becomes,through substitution, a different functional group. For example, a“substituted phenyl group” must still comprise the phenyl moiety and cannot be modified by substitution, in this definition, to become, e.g., apyridine ring. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and subbranches, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. Illustrative substituents include, for example, thosedescribed herein. The permissible substituents can be one or more andthe same or different for appropriate organic compounds. For purposes ofthis invention, the heteroatoms such as nitrogen can have hydrogensubstituent and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valencies of the heteroatoms.Examples of substituents include, but aren't limited to, halogen, azide,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino,nitro, sulfhydryl, amino, amide, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone,aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties,—CF₃, —CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl,heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, halide, oxo,acylalkyl, carboxy esters, -carboxamido, acyloxy, aminoalkyl,alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino,aralkylamino, alkylsulfonyl, -carboxamidoalkylaryl, -carboxamidoaryl,hydroxyalkyl, haloalkyl, alkylylaminoalkylcarboxy-,aminocarboxamidoalkyl-, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl,and the like.

In other embodiments, this invention also relates to compounds havingMRI, PET, CT, X-ray, SPECT, optical or ultrasound signals and their usein the analysis of the location, presence, ad/or quantity of aparticular target in vivo. In some embodiments, agents or compositionsare provided to be detectable in a MRI, PET, CT, X-ray, SPECT, opticalor ultrasound apparatus. In some cases the MRI, PET, CT, X-ray, SPECT,optical or other signal produced by the agent can be affected by thepresence of a particular target. The use of such agents can facilitatethe study of the physiological roles of species including, for examplezinc (Zn²⁺), copper (Cu²⁺), iron (Fe²⁺ or Fe³⁺) or other targets and canaid in the diagnostic and therapy of pathological conditions.

Another advantage of the present invention relates to the capability tosynthesize a broad variety of agents and compositions with differentsignal detections properties (e.g. MRI, PET, CT, X-ray, SPECT, optical,ultrasound based detection) and designed to specifically interact with aparticular target. For example, the invention can involve the synthesisof an agent that suits several applications. In some cases, the agentcan be bound to a metal ion which determines the mechanism of detectionby which the agent determines the target. In an example embodiment theinvention can comprise the synthesis of an agent which can be used as anoptical based agent. Further binding of the same molecular unit to aparamagnetic or radioactive metal ion can then produce an MRI, PET, CT,X-ray, SPECT based agent. In another variation the molecular platformbound to a paramagnetic metal might be used as an optical and MRI agent.Depending on the paramagnetic metal the molecular platform can be usedas a T1, T2 and/or PARACEST contrast agent. The molecular platform canalso comprise one or more attachment sites for various binding moietiesselected to specifically interact with a target.

In some embodiments, the present invention provides methods fordetermination of a target, such as Zn²⁺, Cu²⁺ and/or other biologicaltargets. The agent or composition can comprise a chelator group havingan MRI, PET, CT, X-ray, SPECT, optical or ultrasound signal and abinding moiety covalently attached to the chelator group for binding atarget.

The localization and determination of the target such as Zn²⁺ in vivo isbased on the localization of the agent as total signal or by time-courseanalysis of the agents' retention in a particular site of the body.Alternatively, determination of the target can comprise exposing theagent or composition to a sample suspected containing the target. Ifpresent, the target can interact with the agent to produce a particularMRI, PET, CT, X-ray, SPECT, optical or ultrasound signal of the agent orcomposition.

In some embodiments, the present invention provides methods for imagingparticular locations within a living organism. The agent or compositioncomprise of a chelator group having an MRI, PET, CT, X-ray, SPECT,optical or ultrasound signal and a binding moiety covalently attached tothe chelator group. Where the agent or composition presents a preferredaccumulation in a particular location within a living organism, whichallows the detection of a signal by MRI, PET, CT, X-ray, SPECT, opticalor ultrasound apparatus. In some embodiments such accumulation might bedue to interaction of the agent or composition with a target. In anotherembodiment the accumulation can be directed by the interaction of theagent or composition with another molecule different to the target giventhat the agent or composition might also interact with the target.

As described herein agents or compositions can comprise a chelator groupand a binding moiety covalently attached to the chelator group. Thechelator group can be capable of binding a metal ion such as aparamagnetic or radioactive metal ion and the binding moiety can becapable of binding a target. As described herein, the presence of ametal ion bound to the chelator group, or the type of metal ion bound tothe chelator group, can affect one or more properties of the agent, suchas an MRI, PET, CT, X-ray, SPECT, optical or ultrasound property. Insome cases, the agent can be capable of binding a target and thepresence of the target can affect one or more properties of the agent.In some cases, when the agent binds the target it can bind anothermolecule (for example human serum albumin) and the presence of thesemolecule can affect one or more properties of the agent.

The agents described herein bind to a target for instance Zn²⁺ and thencould associate and/or bind with the ubiquitous serum albumin to form acomplex and with increased longitudinal relaxivity, become more usefulfor MRI. The association of the agent with the relevant metals willallow for the delineation and localization of the sites where the targetmetal is released, retarded, accumulated or trapped, thus allowing forMRI, PET, SPECT or optical detection depending on the composition of themetal center of the contrast agent, whether MRI active, opticallytraceable, radioactive or not.

In some cases, the chelator group comprises of three or more heteroatomswithin a cyclic structure herein called macrocycle, which can coordinatea metal ion as shown in FIGS. 1, 2, 3 and 4. The heteroatoms can benitrogen, oxygen, sulfur, etc. with nitrogen being preferred. In someembodiments, the chelator comprises at least three, at least fourheteroatoms or greater. In some cases as depicted in FIGS. 3 and 4, thechelator group contains at least one cycle within the macrocycle.

Some agents or compositions of the invention can further comprise abinding moiety covalently attached to the chelator capable of binding atarget. In some cases, the binding moiety might comprise one or moregroups capable of interacting with the target which in combination mightfurther interact with another molecule. In some embodiments, the bindingmoiety can comprise one or more heteroatoms capable of binding a metalion or other species present with a living organism (e.g. animal,human). In some embodiments, the binding unit is N,N′-bis-2-pyridylmethyl amine (BPMA).

Some embodiments of the invention comprise an agent having the formulashown in FIG. 1 and FIG. 2, wherein: X₁, X₂, X₃ and X₄ can be the sameor different and each is a heteroatom preferably nitrogen. M can beabsent or if present M is a metal ion which could be used in MRI, PET,SPECT and optical, depending on the relaxometric, luminescent,fluorescent, or radioactive properties of the final form of the metalchelate contrast agent before and after binding to the relevant target.For example for diagnostic by MRI Gd³⁺, Eu³⁺, Tm³⁺, Dy³⁺, Tb³⁺, Yb³⁺ andMn²⁺. For radiodiagnostic ¹¹¹In³⁺, ^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺,^(99m)Tc⁴⁺, ⁶⁴Cu²⁺, Tl3+, or, for radiotherapy ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺ and²¹²Bi³⁺, and ⁹⁰Y³⁺, ³⁷⁷Lu³⁺ and ²²⁵Ac³⁺. Other metal ions forradiodiagnostic or radiotherapeutic applications include: ¹⁴⁹Pm, ¹⁵⁹Gd,¹⁴⁰La, ¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu,¹⁹⁸Au, ^(117m)Sn, and ²¹²Pb. Other metals can be Co, Pr, Nd, Pm, Sm, Ho,Er, Lu and Y. n₁, n₂, n₃ and n₄ can be the same or different and eachindicates a number of methylene (CH₂) units within the heterocyclicstructure. These can be one, two, three or greater but preferably one.R₁ and R₂ can be the same or different and each is an acetate grouppreferably, optionally this can be substituted in the methylene group.Alternatively, R₁ and R₂ can be an acetyl amide group which nitrogen canbe substituted by an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl orheterocycle unit optionally substituted. Alternatively, R₁ and R₂ can bean acetyl ester where the oxy substituent can be alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkynyl, heteroalkynyl, aryl or heterocycle unit optionallysubstituted. Alternatively, R₁ and R₂ can comprise an organic compoundhaving binding affinity for a cell surface receptor. Examples of suchunits comprise peptides, peptoids, antibodies. R₃ indicates asubstituent or substituents in the methylene units of the macrocycle.These substituents can be of any chemical nature as described herein andif several they can be the same or different. A₁ and A₂ can be the sameor different and each indicates an acetyl group preferably, optionallythis can be substituted in the methylene group. H₁ and H₂ can be thesame or different and each indicates a heteroatom preferably nitrogen.S₁ and S₂ can be the same or different and each indicates an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, aryl or heterocycle unit optionallysubstituted. Alternatively, S₁ and S₂ might not be present. E₁ and E₂can be the same or different and each indicates a heteroatom preferablynitrogen. Alternatively, E₁ and E₂ might not be present. L₁, L₂, L₃ andL₄ can be the same or different and each comprises a target bindingmoiety which upon binding the target can bind another molecule furtherfor example a protein. L₁, L₂, L₃ and L₄ can be a substituted derivativeof a heterocycle unit such as but not limited to pyridine, pyrazole,imidazole, piperidine, pyrazine, pyrimidine, pyrrol. Alternatively, L₁,L₂, L₃ and L₄ can be an alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl oraryl optionally substituted.

In some embodiments of the invention, the agent has the formula shown inFIG. 2, wherein: B comprises a cyclic compound within the macrocycleincluding one heteroatom of the macrocycle. This cyclic moiety can bealiphatic or aromatic with or without heteroatoms other than the one inthe macrocycle, optionally the cycle might be substituted.

In some embodiments of the invention, the agent has the formula shown inFIG. 3, wherein: C includes a cyclic compound within the macrocycle.This cyclic moiety can be aliphatic or aromatic with or withoutheteroatoms, optionally substituted.

Agents of the present invention can be synthesized using a variety ofknown methods. In some cases, synthesis of an agent can compriseattachment of the chelator group to one or more binding moieties as inE₁-L₁(-L₂), E₂-L₃(-L₄), optionally via a linker comprising the unitsH₁-S₁-E₁-L₁(-L₂), H₂-S₂-E₂-L₃(-L₄) using a coupling reagent. Thechelator group can comprise at least one functional group capable offorming a bond with a binding moiety, or linker for binding moiety.

An important class of chelators included in some embodiments of theinvention are based on tetraaza macrocycle units, for example1,4,7,10-tetraazacyclododecane (cyclen) which are usually functionalizedin the secondary amines with electrophilic compounds such as halidecontaining reagents. However, these processes are deficient giving finalproducts in poor to moderate yields. Among the problems associated whenusing halide containing reagents are intra- or inter aminequaternization when preparing or using halide compounds comprising atertiary amine in their structure such as the case of BPMA unitdiscussed in some embodiments of the present invention. Additionally,incomplete macrocycle secondary amine substitution occurs when bulkyhaloacetylamide derivatives are used. Those of ordinary skill in the artwill understand the meaning of these terms. A way to solve theseproblems from previous states of the art and as an embodiment of thepresent invention is to use macrocycle derivatives which contain theheteroatom preferably nitrogen substituted with an acetate group.Alternatively, some of the carboxylic acids of the acetates, one,preferably two or three might be substituted as described herein, ifforming an ester as described a tert-butyl ester is preferred. Examplescomprise 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetracetic acid (DOTA),DOTA-bis-tert-butyl ester and derivatives as described herein. Anotherembodiment of the invention comprises the direct attachment of thebinding moiety to the acetate units of the chelator. Any conventionalmethod can be used to produce DOTA and derivatives for example thesynthesis of DOTA-bis-tert-butyl ester has been described in the MexicanPatent application GT/a/2004/000018 by L. M. De León-Rodríguez and Z.Kovacs and DOTA synthesis was described by J. F. Desreux, Inorg. Chem.19(5): 1319-24 (1980). Both references incorporated herein.Alternatively, such compounds are commercially available.

The coupling agent can include any conventional compound known by thoseskilled in the art to facilitate the coupling of the binding unit to thechelator. In particular uronium/guanidium or uronium-containing couplingreagents or phosphonic acid cyclic anhydrides can be used. In certainembodiments, for example, the coupling agent is2-(1H-Benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) in either N- or O-form. The term N- or O-form as used herein,refer to the atoms, either nitrogen or oxygen, directly involved informing the covalent linkage between the benzotriazole group and thetetramethyluronium group. In other embodiments, for example, thecoupling agent is 1-propanephosphonic cyclic anhydride. Such compoundsare available from commercial sources.

In one embodiment, the chelator, coupling agent and binding unit arecombined in a solvent capable of dissolving these compounds to form ahomogeneous mixture. It is advantageous for the solvent to be an aproticorganic solvent, such as dimethylformamide. Alternatively, a base can beadded to the homogeneous mixture. Preferred bases are non-nucleophilictertiary amines for example N,N,N′-diisopropylethylamine, trietylamineor alternatively inorganic bases such as alkaline metal carbonates orbicarbonates for example sodium carbonate. The molar ratio chelator,coupling agent, binding unit and alternatively base in the mixture isabout 1:1 times the number of acetate arms in the chelator:1 times thenumber of acetate arms in the chelator:2 times the number of acetatearms in the chelator. The reaction is preferably carried out bymaintaining the solution containing the chelator, coupling agent,binding unit and base at a constant temperature with stirring for about2 to about 24 hours (e.g., about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, or about 24 hours) to allow attachment of thechelator to the binding unit. The reaction can be carried out at anytemperature where the mixture remains in liquid state; from about −10°C. to about 50° C. (e.g., −5° C., 0° C., 5° C., 10° C., 15° C., 20° C.,25° C., 30° C., 35° C., 40° C., 45° C.), or at about room temperature(e.g., about 22° C.).

After the reaction period, the organic solvent is removed byconventional techniques, such as vacuum evaporation at about 40° C. Theresidual reaction mixture is dissolved in an organic solvent, such asdichloromethane and is washed multiple times with water which mightcontain sodium hydroxide about 0.1 M to about 0.3 M. The washing stepsare preferably carried out in a separatory funnel to facilitate removalof the washing solvent.

Next the organic phase is transferred to another vessel and ahydroscopic salt, such as sodium sulfate is added to the vessel toremove residual water. The mixture is filtered, for example in asintered glass filter. The filtrate is collected and solvent removed byconventional techniques. The residual product contains the contrastagent which might be further purified by if needed.

If tert-butyl groups are present in the final product this can beremoved following ordinary procedures. For instance the agent containingtert-butyl groups can be dissolved in HCl 1M in water and stirred atroom temperature for 2 hours. Solvent can then be removed by ordinarytechniques.

FIG. 5 shows an illustrative embodiment of a macrocycle-based agent fordetermining a zinc analyte. The compound includes two BPMA targetbinding units covalently attached to a tetraaza macrocycle the chelator.The synthesis of this agent is shown in FIG. 6 and follows the proceduredescribed in some embodiments of the present invention. In absence of ametal the optical properties of the agent might be modified depending onthe presence or absence of Zn²⁺ the target. By addition of aparamagnetic metal (e.g. Gd³⁺) an agent having an MRI signal can begenerated. In the presence of zinc the BPMA units can bind to the targetenhancing the relaxivity of the complex to generate a target-bound MRIsignal. FIG. 7 shows an illustrative embodiment of the relaxivityproperties of compound in FIG. 5 when the macrocycle chelates Gd³⁺. Therelaxivity increases with increasing amounts of zinc or copper but notwith addition of calcium and magnesium, which indicates that the targetbinding units are selective for zinc and copper.

In another embodiment the invention provides a method to enhance thelongitudinal relaxivity of the agent upon binding to the target (e.g.Zn²⁺) and subsequent binding to serum albumins such as human serumalbumin (HSA) in humans as exemplified in FIGS. 8, 9 and 10.

In another embodiment the invention comprises MRI protocols in which theagent is deemed injected in low enough concentration as not to causecontrast enhancement in the absence of significant analyte, but exhibitcontrast upon target release from cells, tissues or organs. In otherembodiment, analogous PET/PET-CT and SPECT techniques with similarspatial-temporal-based applications of the analogs of the agents and orcompositions described herein are also outlined using the appropriateradionuclides (e.g., ⁶⁴Cu, or ⁸⁶Y for PET and ¹¹¹In, or ^(99m)Tc forSPECT). Attachment of moieties such as dyes, nanoparticles, and quantumdots are also described for optical localization and imaging.

In some embodiments of the invention the agent or compositions describedherein allow to image localized sites of the target, e.g., Zn²⁺ releasein the pancreas upon glucose-stimulated insulin release (GSIS). Zinc, asfree ions is released into the extracellular space of the beta cell inthe pancreatic islets of Langerhans with insulin release. Such a releaseof insulin is, therefore, correlated with the release of Zn²⁺ that isimaged with the contrast agent. Sample MRI and SPECT images of theanimal trials are shown in FIGS. 11 and 12. This invention provides away to image the event of insulin release in the functioning pancreas orpancreatic region.

Some embodiments of the present invention can advantageously provideagents or compositions that could image the accumulation of Zn²⁺ indiseased states such as Alzheimer's disease in analogs that could crossthe blood-brain barrier (e.g., with the right hydrophobicity index.

For example, an agent as described herein can provide a method ofmonitoring the levels of the target (e.g., Zn²⁺) in the progression of adisease such as diabetes and Alzheimer's.

In some embodiments the invention provides a method of monitoring thelevels of the target at extremely low administration concentration ofthe agent or composition such as in the micromolar range for MRI andnanomolar or lower with other nuclear imaging techniques that involvingradionuclides (e.g., PET, SPECT).

In another embodiment the present invention can provide a process forthe preparation of an agent of formula shown in FIGS. 1, 2, 3 and 4,said process including (i) an organic compound having binding affinityfor an cell surface receptor to (ii) facilitate the retention of thecontrast agent close to or near where the target metal ion is releasedor accumulating. The receptor must be detectable in an in vivodiagnostic imaging procedure; and the linker must couple vector toreporter, at least in significant degree until the imaging procedure hasbeen completed.

In another embodiment the present invention can provide a process forthe preparation of an agent formulation that will allow for themonitoring of the agent retention, retardation or accumulation withultrasound. When the target (e.g., Zn²⁺) binding domains are preservedand the ligand is connected to an ultrasound-visible entities such asmicrobubbles (or other particles with enhanced echogenicity), theinvention allows for the localization of the sites that release orretain the target (e.g., Zn²⁺).

In one embodiment, the agent can be a compound which does not elicit anyunacceptable biological response, particularly compounds that act tosequester enzyme-bound or nucleic-acid bound metal ions and factors thatcause blood-pressure modifying responses at the administrationconcentration. However, biological responses can, if desired, bemodified by administration of a therapeutic agent, e.g., before, duringor after administration of the agent of formula shown in FIGS. 1, 2, 3and 4.

EXAMPLES

¹H- and ¹³C-NMR spectra were recorded on a Varian Gemini: Unity plus 200MHz.

High resolution spectra were recorded on a Varian INOVA 400 MHzspectrometer. Data are reported in the following order, chemical shiftin ppm (δ); muliplicities are indicated as b (broadened), s (singlet), d(doublet), t (triplet), q (quartet), m (multiplet); coupling constants,J, are reported in Hz; integration is provided. Mass spectra (MS) weremeasured either with a HPLC/ES-MS spectrometer Agilent 1100 SeriesLC/MSD trap or a Voyager-DE PRO BioSpectrometry Workstation (MALDI-TOF)]operating in reflector mode using α-cyano-4-hydroxycinnamic acid as thematrix. HPLC purification was performed on a Hewlett Packard Series 1050system using a Jupiter 10μ C18 300A 10 mm×250 mm column. MR images wereobtained using a 400-MHz (9.4 T) vertical-bore Varian INOVA microimagingsystem. T1's were measured using a Maran Ultra NMR relaxometer operatingat 23 MHz.

Relaxivity measurements. Longitudinal relaxivity values were determinedfrom the slope of the line of the reciprocal of T1 versus theconcentration of gadolinium. A 5 mM solution of the gadolinium complexwas made up in Tris buffer 0.1 M pH 7.6 or in buffer plus HSA 600 μM ormale human blood serum. These were serially diluted four times to givefive different sample concentrations at a [Gd]:[Zn] ratio of 1:0. ZnCl₂(CuCl₂) was added to each of the samples to give a [Gd]:[Zn] ratio of1:0.5. After 30 min of incubation at 37° C., T1 measurements were made.This titration was repeated until a 1:3 ([Gd]:[Zn]) ratio was reached.Similar titration experiments were done with CaCl₂ and MgCl₂ to test theeffects of competing cations.

MRI. In vitro phantom MR images were obtained of GdDOTA-diBPMA 0.1 mM inTris buffer 0.1 M pH 7.6 plus HSA 600 μM, with 30, 100 and 200 μM Zn²⁺loaded in 1 mm capillary tubes. Images and T1 values were obtained on a400-MHz (9.4 T) vertical-bore Varian INOVA microimaging system using aspin-echo multislice (SEMS) sequence. The following parameters wereused: Repetition time (TR)=200.0 ms; echo time (TE)=8.3 ms; FOV 15×15mm², data matrix=128×128. No average. A single slice of 5 mm wasacquired centered at the sample height (10 mm). Image analyses werecarried out using ImageJ 1.41o software provided by the NationalInstitutes of Health, USA.

Determination of GdDOTA-Zn₂diBPMA-HSA dissociation constant byrelaxometric demonstrations. HSA was titrated by adding increasingamounts of a GdDOTA-diBPMA 0.1 mM solution in Tris buffer 0.1 M pH 7.6and the T1 values recorded.

Some embodiments of the present invention are also described in DeLeon-Rodriguez et al. J. AM. CHEM. SOC. 2009, 131, 11387-11391. Theentire content is herein incorporated by reference in its entirety. Morespecifically, additional embodiments are incorporated by reference;none-limiting examples include modifications of the novel contrastagent, compound characterization, methods of use thereof, detailedpreparation procedures, and in vivo and in vitro experimental resultsare incorporated herein in their entirety.

Example 1

1,4,7,10-tetraazacyclododecane-1,4-bis(N,N-bis(2-pyridylmethyl)aminoethyleneacetamide)-7,10-aceticacid, DOTA-diBPMA. To a solution of1,4,7,10-Tetraazacyclododecane-1,4-bis(tert-butyl acetate)-7,10-aceticacid (166 mg, 0.321 mmol) in DMF (10 ml) was added a solution of HBTU(243 mg, 0.642 mmol) in DMF (5 mL). The mixture was stirred for 15minutes at room temperature followed by slow addition of a solution ofN,N-bis(2-pyridyl-methyl) ethylene diamine (BPMA) (155 mg, 0.642 mmol)in DMF. The mixture was stirred at room temperature overnight. Thesolvent was evaporated and the residue was taken into CHCl₃ (20 mL) andwashed with deionized water (1×10 mL) and then extracted to the aqueousphase with acetic acid 1M (3×20 mL). The collected aqueous phases werethen basified to pH 13 and the product extracted with CHCl₃ (3×20 mL).The combined organic phases were dried with anhydrous K₂CO₃ and thesolvent was removed by rotary evaporation yielding an orange oil (230mg, 74%). ¹H NMR (200 MHz, CDCl₃; TMS): δ 1.32 (18H, s, tbut), 2.2-3.2(32H, bm, —CH₂—), 3.84 (8H, s, N—CH₂-py), 7.15 (4H, t, 5-H,³J_(HH)=6.4), 7.31 (4H, d, 3-H, ²J_(HH)=7.7), 7.63 (4H, t, 4-H,³J_(HH)=7.6), 8.52 (4H, d, 6-H, ²J_(HH)=4.6). ³C NMR (200 MHz, CDCl₃,TMS): δ 28.2, 50.2, 53.0, 55.8, 56.6, 60.1, 77.4, 81.6, 122.4, 123.45,136.9, 149.1, 159.3, 171.1, 171.8. MS (ESI-positive) m/z=966 [M+H]+(calcld 966).

A solution of the previous compound (60 mg) in CH₂Cl₂, was added dropwise a solution of trifluoroacetic acid (1.5 mL) in CH₂Cl₂ (2 mL). Theresultant solution was stirred for 2 hours and then the solvent wasremoved by rotary evaporation. The residue was washed with cold ethylether (3×10 mL) and then was taken into deionized water with HCl 0.1 M(10 mL) and washed with CHCl₃ (3×10 mL). The aqueous phase was freezedried yielding a highly hygroscopic orange solid (quant.). ¹H NMR (200MHz, D2O): δ 2.6-3.5 (28H, bm, —CH₂—), 3.67 (4H, s, —CH₂—), 3.85 (8H, s,N—CH₂-py), 7.25 (4H, t, 5-H, ³J_(HH)=6.8), 7.38 (4H, d, 3-H,²J_(H)H=7.9), 7.73 (4H, t, 4-H, ³J_(HH)=7.5), 8.33 (4H, d, 6-H,²J_(HH)=4.9). ¹³C{1H}NMR (50 MHz, D₂O): 36.8, 48.0, 51.1, 53.0, 55.3,56.3, 59.2, 123.1, 124.4, 138.1, 147.7, 156.7, 169.0, 171.7. MS(ESI-positive) m/z=826 [M+H]+ (calcld 826).

General procedure for Gd³⁺ and other metal ions DOTA-diBPMA synthesis.DOTA-diBPMA was dissolved in MilliQ grade H₂O and the pH adjusted to 6.5with 1 M NaOH. GdCl₃.6H₂O in slight excess was slowly added. The pH ofthe solution was maintained between 6 and 6.5 during addition. Resultantsolution was stirred at room temperature for several days and pHadjusted close to 6.5 as needed. Unreacted Gd³⁺ was precipitated asGd(OH)₃ after the addition of 1 M NaOH and the crude mixture waspurified by semi-preparative HPLC with aqueous (NH₄)₂CO₃ 5 mM pH 8/MeOH(50/50) as the eluant. The complex was obtained as a yellow solid. m/z(MALDI+) m/z=1008.67 [M+H]+ (calcld. 1008.26). Anal. Calcd forC₄₄H₅₈GdN₁₂O₆.2H₂O: C, 50.61; H, 5.98; N, 16.10. Found: C, 50.81; H,5.96; N, 16.18

Example 2

The following example describes the use of GdDOTA-diBPMA in the in vivoimaging of Zn²⁺ by MRI. GdDOTA-diBPMA was shown to bind to Zn²⁺ tightlyand as such bind to serum albumin. For this study, rodents (rats andmice) were fasted overnight (12-16 hours) before treatment. Thereafter,an intraperitoneal (i.p.) injection of 20% glucose solution was given,then a solution of GdDOTA-diBPMA was injected intravenously (e.g., tailvein) within a 20 minute window as a bolus. The dynamic contrastenhancement (DCE) was monitored prior to until after 30 min to 2 hrs ofthe i.p. injection of glucose and the administration of the contrastagent. The T1-based contrast enhancement that was observed only inanimals injected with glucose versus normal saline solution as controlshowed the sites (e.g., in the pancreas) that release Zn²⁺ ions (FIG.11).

Example 3

The following example describes the use of GdDOTA-diBPMA in the in viveimaging of Zn²⁺ by MRI as applied to delineate diabetic versus normalphysiology. In this example, the presence or absence of the contrastenhancement is directly correlated with the presence or absence ofinsulin-releasing sites in the pancreas or zinc containing, retaining orreleasing sites in other parts of the body. The same fasting method andimaging was done for animals as in Example 2 above. The animals wereinduced to be type I diabetic by treatment with the antibioticstreptozotocin for 5 days and imaged by MRI after testing to bysymptomatic of diabetes. The MR imaging protocol used for these animalswere the same as the ones used on them prior to being diabetes (e.g.,glucose by i.p. injection and compound 1 treatment prior to T1-weightedDCE). The differences in contrast enhancement, e.g., the generallowering and/or disappearance of T1-weighted contrast enhancement fromthe normal to diabetic state, was observed both for individual animalsand as a general trend. The disappearance of localized andMRI-observable T1-enhancements (as observed in FIG. 11B) in the pancreaswas correlated with the disappearance of functioning beta-cellcontaining islets of Langerhans in the pancreas. The beta cells, whichrelease the insulin, are know to co-release zinc ions into theextracellular space at the same time upon glucose-stimulated insulinsecretion.

Example 4

The following example describes the use of compound ⁶⁴Cu²⁺ DOTA-diBPMA(analog of GdDOTA-diBPMA) in the in vive imaging of Zn²⁺ by PET. Thesame overnight fasting treatment and imaging of the animals prior toGSIS and intravenous contrast agent administration was done as inExample 2. The PET imaging of the animals, both pre- and post-diabeticstates, was done prior to, during and after contrast agent injection. Itwas observed that the fasted animals that were not treated or stimulatedwith glucose did not show pronounced contrast enhancement, unlike in theglucose-stimulated animal group which showed numerous sites in thepancreas that showed signal enhancement with PET. It was also observedthat the animals lost significant localized pancreas enhancement afterstreptozotocin treatment (i.e., diabetic state) when given the same PETimaging treatment protocol (FIG. 12).

Example 5

The following example describes the use of GdDOTA-diBPMA for MRI or itsPET analog (⁶⁴Cu²⁺ DOTA-diBPMA) for mapping the Zn-binding capacity ofan animal. Low blood levels of the GdDOTA-diBPMA (sub-millimolar) or itsPET analog (⁶⁴Cu²⁺ DOTA-diBPMA; at nanomolar or sub-nanomolar levels)can be injected directly into the bloodstream to map the parts of thebody that can have enough of the metal ion that can be chelated andbound to serum albumin and followed by dynamic scanning by MRI and PET,respectively. The T1-enhancement that develops during the blood lifetimeof the agent(s) allows for the imaging of normal or diseased tissuesthat can retain, retard or accumulate the agent-bound zinc by MRI. Also,the PET-analog can give a map of cells, tissues or organs that releaseor have available Zn²⁺ ions by PET imaging with or without chemical(e.g., glucose or other substrates) stimulation. Such diseased statesinclude, but are not limited to prostate and other types of cancer,Alzheimer's disease, and diabetes.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification can mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations can be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A MRI, PET, CT, X-ray, SPECT, optical orultrasound agent or composition having formula:

wherein X₁, X₂, X₃ and X₄ independently represent heteroatoms; M is ametal selected from the group consisting of Gd³⁺, Eu³⁺, Tm³⁺, Dy³⁺,Tb³⁺, Yb³⁺, Mn²⁺, ¹¹¹In³⁺, ^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺,⁶⁴Cu²⁺, Tl³⁺, ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺, ²¹²Bi³⁺, ⁹⁰Y³⁺, ³⁷⁷Lu³⁺, ²²⁵Ac³⁺, ¹⁴⁹Pm,¹⁵⁹Gd, ¹⁴⁰La, ¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu,¹⁹⁸Au, ^(117m)Sn, and ²¹²Pb, Ce, Pr, Nd, Pm, Sm, Ho, Er, Lu and Y; n₁,n₂, n₃, and n₄ are each independently a natural integer; R₁ and R₂ eachrepresent an acetyl group or an organic compound having binding affinityfor a cell surface receptor; R₃ represents at least one substituent inthe methylene unit of the macrocycle; A₁ and A₂ independently representacetyl groups; H₁ and H₂ independently represent heteroatoms; S₁ and S₂independently represent an alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,aryl or heterocycle unit or a direct bond; E₁ and E₂ independentlyrepresent heteroatoms; and L₁, L₂, L₃, and L₄ represent a target bindingmoiety capable of binding another molecule upon binding the target, andare independently selected from the group consisting of a substitutedderivative of a heterocycle unit, pyridine, pyrazole, imidazole,piperidine, pyrazine, pyrimidine, pyrrol, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl and aryl substituted group, and wherein the agent orcomposition is capable of detecting a target in vivo in a subject.
 2. AMRI, PET, CT, X-ray, SPECT, optical or ultrasound agent or compositionhaving formula:

wherein X₁, X₂, X₃ and X₄ independently represent heteroatoms; M is ametal selected from the group consisting of Gd³⁺, Eu³⁺, Tm³⁺, Dy³⁺,Tb³⁺, Yb³⁺, Mn²⁺, ¹¹¹In³⁺, ^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺,⁶⁴Cu²⁺, Tl³⁺, ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺, ²¹²Bi³⁺, ⁹⁰Y³⁺, ³⁷⁷Lu³⁺, ²²⁵Ac³⁺, ¹⁴⁹Pm,¹⁵⁹Gd, ¹⁴⁰La, ¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu,¹⁹⁸Au, ^(117m)Sn, ²¹²Pb, Ce, Pr, Nd, Pm, Sm, Ho, Er, Lu and Y; n₁, n₂,n₃, and n₄ are each independently a natural integer, R₁ represents anacetyl group or an organic compound having binding affinity for a cellsurface receptor; R₃ represents at least one substituent in themethylene unit of the macrocycle; A₁ and A₂ independently representacetyl groups; H₁ and H₂ independently represent heteroatoms; S₁ and S₂independently represent an alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,aryl or heterocycle unit or a direct bond; E₁ and E₂ independentlyrepresent heteroatoms; L₁, L₂, L₃, and L₄ represent a target bindingmoiety capable of binding another molecule upon binding the target, andare independently selected from the group consisting of a substitutedderivative of a heterocycle unit, pyridine, pyrazole, imidazole,piperidine, pyrazine, pyrimidine, pyrrol, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl and aryl substituted group; and B represents an aliphaticor aromatic cyclic compound comprising heteroatom X₃, and wherein theagent or composition is capable of detecting a target in vivo in asubject.
 3. A MRI, PET, CT, X-ray, SPECT, optical or ultrasound agent orcomposition having formula:

wherein X₁, X₂, X₃ and X₄ independently represent heteroatoms; M is ametal selected from the group consisting of Gd³⁺, Eu³⁺, Tm³⁺, Dy³⁺,Tb³⁺, Yb³⁺, Mn²⁺, ¹¹¹In³⁺, ^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺,⁶⁴Cu²⁺, Tl³⁺, ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺, ²¹²Bi³⁺, ⁹⁰Y³⁺, ³⁷⁷Lu³⁺, ²²⁵Ac³⁺, ¹⁴⁹Pm,¹⁵⁹Gd, ¹⁴⁰La, ¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu,¹⁹⁸Au, ^(117m)Sn, ²¹²Pb, Ce, Pr, Nd, Pm, Sm, Ho, Er, Lu and Y; n₁, n₂,and n₃ are each independently a natural integer; R₁ and R₂ independentlyrepresents an acetyl group or an organic compound having bindingaffinity for a cell surface receptor; R₃ represents at least onesubstituent in the methylene unit of the macrocycle; A₁ and A₂independently represent acetyl groups; H₁ and H₂ independently representheteroatoms; S₁ and S₂ independently represent an alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, aryl or heterocycle unit or a direct bond;E₁ and E₂ independently represent heteroatoms; L₁, L₂, L₃, and L₄represent a target binding moiety capable of binding another moleculeupon binding the target, and are independently selected from the groupconsisting of a substituted derivative of a heterocycle unit, pyridine,pyrazole, imidazole, piperidine, pyrazine, pyrimidine, pyrrol, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl and aryl substituted group; and Crepresents an aliphatic or aromatic cyclic compound within themacrocycle, and wherein the agent or composition is capable of detectinga target in vivo in a subject.
 4. A MRI, PET, CT, X-ray, SPECT, opticalor ultrasound agent or composition having formula:

wherein X₁, X₂, and X₃ independently represent heteroatoms; M is a metalselected from the group consisting of Gd³⁺, Eu³⁺, Tm³⁺, Dy³⁺, Tb³⁺,Yb³⁺, Mn²⁺, ¹¹¹In³⁺, ^(113m)In³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ^(99m)Tc⁴⁺, ⁶⁴Cu²⁺,Tl³⁺, ¹⁵³Sm³⁺, ¹⁶⁶Ho³⁺, ²¹²Bi³⁺, ⁹⁰Y³⁺, ³⁷⁷Lu³⁺, ²²⁵Ac³⁺, ¹⁴⁹Pm, ¹⁵⁹Gd,¹⁴⁰La, ¹⁷⁵Yb, ⁴⁷Sc, ¹⁸⁶Re, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁷Pt, ⁶⁷Cu, ¹⁹⁸Au,^(117m)Sn, and ²¹²Pb, Ce, Pr, Nd, Pm, Sm, Ho, Er, Lu and Y; n₁, n₂, andn₃ are each independently a natural integer; R₁ represents an acetylgroup or an organic compound having binding affinity for a cell surfacereceptor; R₃ represents at least one substituent in the methylene unitof the macrocycle; A₁ and A₂ independently represent acetyl groups; H₁and H₂ independently represent heteroatoms; S₁ and S₂ independentlyrepresent an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl orheterocycle unit or a direct bond; E₁ and E₂ independently representheteroatoms; and L₁, L₂, L₃, and L₄ represent a target binding moietycapable of binding another molecule upon binding the target, and areindependently selected from the group consisting of a substitutedderivative of a heterocycle unit, pyridine, pyrazole, imidazole,piperidine, pyrazine, pyrimidine, pyrrol, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl and aryl substituted group, and wherein the agent orcomposition is capable of detecting a target in vivo in a subject. 5.The agent or composition according to claim 1, wherein the target is ametal ion or a macromolecular receptor.
 6. The agent or compositionaccording to claim 1, wherein the target is Zn²⁺ or Cu²⁺.
 7. The agentor composition according to claim 2, wherein the target is a metal ionor a macromolecular receptor.
 8. The agent or composition according toclaim 2, wherein the target is Zn²⁺ or Cu²⁺.
 9. The agent or compositionaccording to claim 3, wherein the target is a metal ion or amacromolecular receptor.
 10. The agent or composition according to claim3, wherein the target is Zn²⁺ or Cu²⁺.
 11. The agent or compositionaccording to claim 4, wherein the target is a metal ion or amacromolecular receptor.
 12. The agent or composition according to claim4, wherein the target is Zn²⁺ or Cu²⁺.